Nicotine-induced changes of brain ß-endorphin.

A consensus has emerged that endogenous opioid peptides and their receptors play an important role in the psychoactive properties of nicotine. Although behavioral studies have shown that ß-endorphin contributes to the rewarding and emotional effects of nicotine, whether the drug alters the function of brain endorphinergic neurons is not fully explored. These studies investigated the effect of acute, 1mg/kg, sc, and chronic, daily injection of 1mg/kg, sc, for 14 days, administration of free base nicotine on brain ß-endorphin and its precursor proopiomelanocortin (POMC). Acute and chronic treatment with nicotine decreased ß-endorphin content in hypothalamus, the principal site of ß-endorphin producing neurons in the brain, and in the endorphinergic terminal fields in striatum and hippocampus. The acute effect of nicotine on ß-endorphin was reversed by the nicotinic antagonist mecamylamine and the dopamine antagonist haloperidol, indicating pharmacological specificity and involvement of dopamine D2-like receptors. Similar observations were made in prefrontal cortex. POMC mRNA in hypothalamus and prefrontal cortex was unchanged following acute nicotine, but it decreased moderately with chronic treatment. The nicotine treatments had no effect on pituitary and plasma ß-endorphin. Taken together, these results could be interpreted to indicate that nicotine alters the synthesis and release of ß-endorphin in the limbic brain in vivo. Altered endorphinergic function may contribute to the behavioral effects of acute and chronic nicotine treatment and play a role in nicotine addiction.

Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated phase II enzyme gene expression.

Green tea polyphenols, major constituents of green tea, are potent chemopreventive agents in a number of experimental models of cancer in animals. The mechanisms of cancer protection by these agents are not clear, but may involve modulation of the enzyme systems responsible for the detoxification of chemical carcinogens. The present studies show that a green tea polyphenol extract (GTP) induces chloramphenicol acetyltransferase (CAT) activity in human heptoma HepG2 cells transfected with a plasmid construct which contains an antioxidant-responsive element (ARE) and a minimal glutathione S-transferase Ya promoter linked to the CAT reporter gene. This indicates that GTP stimulates the transcription of Phase II detoxifying enzymes through the ARE. To explore the upstream signaling pathways leading to gene expression, we studied the involvement of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1). Potent activation of ERK2 was seen following treatment of HepG2 cells with different concentrations of GTP. Similar to ERK2, JNK1 was also strongly activated by treatment with GTP, although to a lesser extent and in a different dose-dependent fashion. Kinetic studies revealed that GTP activation of JNK1 was delayed and sustained, whereas ERK2 activation was rapid and transient. Furthermore, GTP treatment also increased mRNA levels of the immediate-early genes c-jun and c-fos, as determined by reverse transcriptase-coupled polymerase chain reaction. Taken together, these studies provide insights into the action of GTP and suggest that the stimulation MAPKs may be the potential signaling pathways utilized by GTP to activate ARE-dependent genes.

Chronic blockade of opioid receptors alters the binding of [3H]GBR 12935 to dopamine transporter in rat brain regions and spinal cord.

The effect of chronic administration of naltrexone, an opioid receptor antagonist, on the activity of the dopamine transporter in brain regions and spinal cord was determined. Male Sprague-Dawley rats were implanted with a pellet containing 10 mg naltrexone for 7 days. Rats serving as controls were implanted with a placebo pellet. Two groups of rats were used. In one, the pellets were left intact, and in the other they were removed 16 h prior to sacrificing. The dopamine transporter was labeled with [3H]GBR 12935. The binding of [3H]GBR 12935 in rats in which naltrexone pellets were left intact was decreased by 63 and 31% in corpus striatum and spinal cord, respectively, when compared to placebo pellet implanted controls. The decrease in binding in the striatum was due to changes in the Bmax value of [3H]GBR 12935; the Kd values did not differ. In hypothalamus, pons-medulla, hippocampus, midbrain, cortex, and amygdala of naltrexone and placebo pellet implanted rats, the binding of [3H]GBR 12935 did not differ. In naltrexone-treated rats from which pellets had been removed, the binding of [3H]GBR 12935 was decreased in hippocampus, amygdala, and spinal cord by 68, 77, and 61%, respectively, in comparison with tissues from the control rats. The results indicate that chronic blockade of opioid receptors results in downregulation of dopamine transporter in selected brain regions and spinal cord.

Modification of the characteristics of dopamine transporter in brain regions and spinal cord of morphine tolerant and abstinent rats.

The specific binding of [3H]GBR 12935 to crude synaptosomal membranes of brain regions and spinal cord of morphine tolerant and abstinent rats was investigated. Male Sprague-Dawley rats were implanted with 6 morphine pellets each containing 75 mg of morphine base during a 7-day period. Placebo pellet implanted rats served as controls. Rats sacrificed without removal of the pellet were considered tolerant whereas those from which pellets were removed 16 hr prior to sacrificing were labeled abstinent. The binding of [3H]GBR 12935 was initially determined at a 1 nM concentration in all brain regions and spinal cord, which was followed by the determination of Bmax and Kd values in the corpus striatum, a highly enriched region for the dopamine transporter. In morphine tolerant rats, the binding of [3H]GBR 12935 was increased in the hypothalamus (182%) but was decreased in the corpus striatum (34%) and spinal cord (30%). The decrease in binding in the corpus striatum was due to an increase in the Kd value of [3H]GBR 12935. However, during morphine withdrawal, the binding of [3H]GBR 12935 was still higher in the hypothalamus (255%) but was decreased in the hippocampus (53%). Thus, chronic administration of morphine results in changes in the dopamine transporter function in selected brain regions and the spinal cord, and these changes are dependent upon whether or not the animals are undergoing the abstinence syndrome.

Differential binding of [3H]MK-801 to brain regions and spinal cord of mice treated chronically with morphine.

1. The effects of morphine tolerance and abstinence on the binding of [3H]MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptors were determined in brain regions and spinal cord of the mouse. 2. Male Swiss-Webster mice were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of a pellet containing 75 mg of morphine base for 3 days. Placebo pellet-implanted mice served as controls. In tolerant (nonabstinent) mice, the pellets were left intact at the time of sacrificing whereas, in the abstinent mice, the pellets were removed 6 hr prior to sacrificing. 3. The binding of [3H]MK-801 to membranes prepared from spinal cord and brain regions (cortex, pons-medulla, hypothalamus, hippocampus, amygdala, striatum, and midbrain) was determined by using a 5 nM concentration of the ligand in the presence of 30 microM glycine and 50 microM of glutamate. 4. In nonabstinent morphine-tolerant mice, the binding of [3H]MK-801 was decreased in pons-medulla and hypothalamus, but was increased in the spinal cord in comparison to that in placebo controls. The reduction in binding in pons-medulla was due to a decrease in the Bmax value; the Kd value remained unchanged. The binding of [3H]MK-801 was increased in the hippocampus of morphine-abstinent mice. 5. These studies demonstrate that NMDA receptors of brain regions and spinal cord are differentially affected in morphine-tolerant and abstinent mice.

Effects of acute and chronic administration of dizocilpine on the pharmacological responses to U-50,488H and brain and spinal cord kappa-opioid receptors in the rat.

In male Sprague-Dawley rats, acute and chronic effects of dizocilpine (MK-801), a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, were determined on the analgesic and hypothermic actions of U-50,488H, a kappa-opioid receptor agonist. In addition, the in vitro effects of MK-801 on the binding of [3H]ethylketocyclazocine ([3H]EKC) to kappa-opioid receptors in brain and spinal cord of the rat were determined. A single injection of MK-801 given 10 min prior to U-50,488H or given twice a day for 4 days dose-dependently enhanced the analgesic action of U-50,488H. The enhancement of the analgesic response was much greater in rats injected chronically with MK-801 as compared with those injected acutely. Both single and multiple injections of MK-801 failed to affect the hypothermic action of U-50,488H. In vitro, MK-801 inhibited the binding of [3H]EKC to brain and spinal cord membranes with IC50 values of 9.80 +/- 1.7 and 1.37 +/- 0.58 microM, respectively. Chronic administration of MK-801 twice a day for 4 days increased the Bmax value of [3H]EKC binding in the brain, but had no effect on Kd. On the other hand, chronic treatment with MK-801 decreased the Kd of [3H]EKC binding in spinal cord without affecting Bmax. It is concluded that blockade of NMDA receptor enhances the analgesic response to a kappa-opioid receptor agonist and upregulates brain and spinal cord kappa-opioid receptors. Finally, the results suggest that the NMDA receptor may have a role in the regulation of kappa-opioid systems.

Preproenkephalin mRNA and methionine-enkephalin content are increased in mouse striatum after treatment with nicotine.

A single dose of nicotine increased methionine-enkephalin (Met-Enk) immunoreactivity in the striatum of mice in a time-dependent manner. Met-Enk content reached maximum by approximately 1 h after nicotine and returned to control values by 6 h. The response to nicotine was blocked by pretreating animals with the nicotinic receptor antagonist mecamylamine. In contrast, pretreating mice with the muscarinic receptor antagonist atropine or the dopamine receptor antagonist haloperidol did not block the response. A single dose of nicotine also increased mRNA for the precursor peptide preproenkephalin (PPE). The increase of PPE mRNA preceded that of Met-Enk and reached a maximum by approximately 30 min after nicotine. PPE mRNA levels returned to near normal by approximately 3 h and increased again by 6 h after nicotine. Daily administration of nicotine for 14 days increased Met-Enk content and PPE mRNA in the striatum of mice as well. Taken together, our results suggest that nicotinic receptors modulate Met-Enk content and PPE mRNA in the mouse striatum.

Modulation of preproenkephalin mRNA levels in brain regions and spinal cord of rats treated chronically with morphine.

The effect of morphine tolerance/dependence and abstinence on the preproenkephalin (PPE) gene expression was determined in brain regions and spinal cord of the rat. Male Sprague-Dawley rats were rendered tolerant and physically dependent on morphine by SC implantation of six pellets, each containing 75 mg of morphine base, during a 7-day period. Placebo pellet-implanted rats served as controls. In tolerant rats, the pellets were left in place at the time of sacrifice whereas in abstinent rats, the pellets were removed 16 h prior to sacrificing. The levels of PPE mRNA were determined in brain regions (striatum, cortex, pons-medulla, hypothalamus, amygdala, and midbrain) and spinal cord. The levels of PPE mRNA increased significantly in the cortex (62%) and the spinal cord (352%) of morphine-tolerant rats when compared to placebo pellet-implanted control rats. In other brain regions, the levels of PPE mRNA in placebo and morphine-tolerant rats did not differ. On the other hand, in morphine-abstinent rats, the levels of PPE mRNA increased in the striatum (62%) and hypothalamus (34%) but were decreased in pons-medulla (68%), midbrain (51%), and spinal cord (36%) in comparison to the placebo controls. The results clearly demonstrate differential changes in enkephalin gene expression in brain regions and spinal cord of the abstinent and nonabstinent morphine-tolerant/dependent rats.

Down-regulation of N-methyl-D-aspartate (NMDA) receptors of brain regions and spinal cord of rats treated chronically with morphine.

1. The effects of morphine tolerance and abstinence on the characteristics of N-methyl-D-aspartate (NMDA) receptors, labeled with [3H]MK-801, were determined in the brain regions and spinal cord of the rat. 2. Male Sprague-Dawley rats were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of six morphine pellets during a 7-day period. In tolerant (non-abstinent) rats, the pellets were left intact at the time of sacrificing, whereas in the abstinent rats the pellets were removed 16 hr prior to sacrificing. 3. The binding of [3H]MK-801, an NMDA receptor antagonist, to membranes prepared from spinal cord and brain regions (cortex, striatum, amygdala, hippocampus, hypothalamus, midbrain and pons-medulla) was determined using 5 nM concentration of the ligand in the presence of 30 microM glycine and 50 microM of glutamate. 4. In non-abstinent morphine tolerant rats, the binding of [3H]MK-801 was decreased by 40 and 33% in the midbrain and spinal cord, respectively, in comparison with their placebo controls. In morphine abstinent rats, the binding of [3H]MK-801 was decreased by 42, 29 and 50% in hypothalamus, midbrain and spinal cord, respectively, in comparison with their placebo controls. The binding of [3H]MK-801 to other brain regions and spinal cord of morphine tolerant and abstinent rats did not differ from their respective placebo controls. 5. Thus, these studies demonstrate, for the first time, that in the presence of glutamate and glycine, NMDA receptors of selected brain regions and spinal cord are down-regulated in rats treated chronically with morphine.

Effect of morphine tolerance and abstinence on the binding of [3H]MK-801 to brain regions and spinal cord of the rat.

The effect of chronic administration of morphine to rats on the N-methyl-D-aspartate (NMDA) receptors labeled with [3H]MK-801, a non-competitive antagonist, was determined in brain regions and spinal cord. Male Sprague-Dawley rats were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of 6 morphine pellets during a 7-day period. Each pellet contained 75 mg of morphine free base. Animals serving as controls were similarly implanted with placebo pellets. This procedure resulted in the development of a high degree of tolerance and physical dependence on morphine. Two sets of rats were used. In one, the pellets were left intact at the time of sacrifice (tolerant) and in the other the pellets were removed 16 h prior to sacrificing (abstinent). The binding constants, Bmax and Kd values of [3H]MK-801 were determined in cortex, hippocampus, hypothalamus, corpus striatum, midbrain and spinal cord. In the absence of glycine and glutamate, [3H]MK-801 bound to tissue membranes at a single high affinity site. The Bmax and Kd values of [3H]MK-801 were not altered in any of the tissues of the morphine abstinent rats. The Bmax value of [3H]MK-801 was significantly decreased in cerebral cortex of morphine tolerant rats as compared to their placebo controls but the Kd values did not change. In other brain regions and spinal cord of morphine tolerant rats and their placebo controls, the Bmax and Kd values of [3H]MK 801 did not differ.(ABSTRACT TRUNCATED AT 250 WORDS)

c-fos and NGFI-A mRNA of rat retina: evidence for light-induced augmentation and a role for cholinergic and glutamate receptors.

When rats are exposed to room light from the dark, there is a transient increase of mRNA for the immediate-early genes c-fos and NGFI-A in the retina. Augmentation of c-fos and NGFI-A mRNA by light is apparently associated with activation of cholinergic nicotinic and muscarinic receptors as it can be suppressed by the nicotinic antagonist mecamylamine and the muscarinic antagonist atropine. Moreover, the light-induced increase of c-fos mRNA in retina appears to be associated with activation of glutamate receptors also as the noncompetitive inhibitor of N-methyl-D-aspartate receptors dizocilpine (MK-801) partially suppressed the increase of the c-fos message. Light-induced NGFI-A mRNA augmentation is apparently modulated by the same receptors. We were unable to detect light-induced changes of c-jun mRNA.

Distribution of aromatic L-amino acid decarboxylase mRNA in mouse brain by in situ hybridization histology.

Aromatic L-amino acid decarboxylase (AAAD) is the second enzyme in the sequence leading to the synthesis of catecholamines or serotonin. Antisense riboprobes for aromatic L-amino acid decarboxylase mRNA were used to map the gene in mouse brain by in situ hybridization. The substantia nigra, the ventral tegmental nucleus, the dorsal raphe nucleus, the locus coeruleus, and the olfactory bulb contained the highest signal for AAAD mRNA. After treatment with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the signal disappeared in the substantia nigra, decreased somewhat in the ventral tegmental area, and remained unchanged in the dorsal raphe nucleus. Hypothalamic and cerebellar Purkinje neurons known to contain histidine decarboxylase or glutamic acid decarboxylase, respectively, were unlabeled by the probes. However, neurons in the deep layers of the frontal cortex, many thalamic nuclei, and the pyramidal neurons of the hippocampus were lightly to moderately labeled for mouse AAAD mRNA. The presence of AAAD message in these neurons suggests that the enzyme has functions other than that for the synthesis of the classical biogenic amine neurotransmitters.

Expression of cloned aromatic L-amino acid decarboxylase in Xenopus laevis oocytes.

Sense mRNA coding for bovine adrenal medulla aromatic L-amino acid decarboxylase (AADC) was expressed following microinjection into Xenopus laevis oocytes. The expressed enzyme activity was stereoselective for L-5-hydroxytryptophan and L-DOPA and blocked by NSD-1015 an inhibitor of AADC. Heating the expressed enzyme at 55 degrees C resulted in a parallel loss of activity towards both substrates. Our findings are consistent with the prevailing notion that a single enzyme is able to decarboxylate both substrates in vivo.

c-fos mRNA in mouse brain after MPTP treatment.

The neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces a transient increase of mRNA for the immediate-early gene c-fos in the mouse brain. The c-fos mRNA level is MPTP dose-dependent and is evident in all brain regions tested including striatum, hypothalamus, cortex, hippocampus, cerebellum and midbrain. There are regional differences in the time-course for the rise of c-fos mRNA. Pretreatment with deprenyl, a selective monoamine oxidase B inhibitor, pargyline, a nonselective monoamine oxidase inhibitor, or mazindol, a dopamine uptake transport inhibitor, does not prevent the c-fos mRNA increase, suggesting that the elevation is due to the action of MPTP and not its neurotoxic metabolite MPP+.

Beta-endorphin and methionine-enkephalin levels in discrete brain regions, spinal cord, pituitary gland and plasma of morphine tolerant-dependent and abstinent rats.

The effect of morphine tolerance-dependence, protracted and naloxone-precipitated abstinence on the levels of beta-endorphin and methionine-enkephalin in discrete brain regions, spinal cord, pituitary gland and plasma was determined in the male Sprague-Dawley rats. Among the brain regions examined, the levels of beta-endorphin in descending order were: hypothalamus, amygdala, midbrain, hippocampus corpus striatum, pons and medulla and cortex. The levels of beta-endorphin in midbrain, hypothalamus, and pituitary of morphine tolerant-dependent rats were decreased significantly. During protracted withdrawal beta-endorphin levels were decreased in amygdala, spinal cord and pituitary. During naloxone-precipitated abstinence beta-endorphin levels were increased in corpus striatum, midbrain and cortex. In addition, in naloxone-precipitated abstinence beta-endorphin levels were decreased in pituitary gland and hippocampus but increased in plasma. The levels of methionine-enkephalin in brain regions in decreasing order were: corpus striatum, pons and medulla, amygdala, hypothalamus, midbrain, hippocampus and cortex. The levels of methionine-enkephalin in pons and medulla, amygdala, hippocampus and pituitary gland were decreased in morphine tolerant-dependent rats. During protracted abstinence from morphine, methionine-enkephalin levels in spinal cord, amygdala, pons and medulla, midbrain, cortex, corpus striatum and pituitary gland were decreased. The levels of methionine-enkephalin in hypothalamus and corpus striatum of naloxone-precipitated abstinent rats were increased but were decreased in amygdala and pituitary gland. These results suggest that during morphine tolerance-dependence and during protracted abstinence beta-endorphin and methionine-enkephalin levels in discrete brain regions and pituitary gland are decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of mu, kappa and delta opioid receptors in the rat brain by isoflurane and enflurane.

This study was done to investigate whether inhalational anesthetics modulated the binding of specific ligands to opioid receptors in the brain of the rat. The effect of isoflurane and enflurane on the binding of specific ligands to various subtypes of opioid receptors in vitro was studied. Isoflurane inhibited the binding of [3H]naloxone to opioid receptors by 50% in the spinal cord, midbrain and cortex at 22, 49 and 50 mM, respectively. Enflurane was more potent than isoflurane in inhibiting the binding of [3H]naloxone. Scatchard analysis of the binding of [3H]naloxone, done in the presence of therapeutic level (5 mM) of isoflurane, suggested that it did not affect the KD (1.3 nM) but decreased the Bmax by 41% in the cortex. Isoflurane and enflurane, at large doses (30-50 mM), inhibited the binding of [3H]ethylketo-cyclazocine (EKC) to kappa receptors in midbrain, cortex and spinal cord. At a smaller dose (5 mM), they increased the binding of EKC in spinal cord. The binding of the analogs of enkephalin [3H]DSTLE(Tyr-D-Ser-Gly-Phe-Leu-Thr-enkephalin) to delta receptors and [3H]DAGO (Tyr-D-Ala-Gly-Methyl-Phe-Glyol-enkephalin) to mu receptors in the midbrain and cortex was inhibited by isoflurane at a significantly smaller concentration than the binding of [3H]naloxone, indicating that the binding of peptides was more susceptible to the inhibition by inhalational anesthetics than the binding of alkaloids, such as naloxone or EKC. These results suggest that the modulation of opioid receptors by inhalational anesthetics is a function of both the nature of the ligand and the tissue used for the receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitation of dimethylbenz[a]anthracene-induced rat mammary tumorigenesis by restraint stress: role of beta-endorphin, prolactin and naltrexone.

In order to investigate the involvement of opioid peptides and prolactin in stress-facilitated mammary cancer, we studied the effect of chronic restraint stress on dimethylbenz[a]-anthracene (DMBA)-induced mammary tumorigenesis and the effect of an opiate antagonist, naltrexone, on this process. Female Fischer-344 rats (n = 160) were administered 15 mg DMBA/ml of sesame oil/rat by intragastric intubation. Eighty rats were subjected to daily 30 min restraint stress in a plastic cylinder, and 80 rats served as control not subjected to the stressor. Half of the rats from each group received naltrexone (1 mg/kg, i.p. daily). Five rats from each group (restraint stress +/- naltrexone and control +/- naltrexone) were killed every 2-3 weeks. Rats subjected to restraint stress developed a greater number of tumors earlier. Naltrexone decreased the tumor incidence in the stressed animals from 32 to 12% (P less than 0.001) and in unstressed rats from 27 to 15% (P less than 0.001) at the end of 18 weeks. Stressed rats showed a decrease of 48% (P less than 0.001) in the level of hypothalamic beta-endorphin. Plasma prolactin increased from 4-13 ng/ml in the control rats to 109-396 ng/ml (P less than 0.001) in the stressed rats throughout the 18 week period. The beneficial effect of naltrexone was associated with 42% (P less than 0.01) increase in T cell proliferation, but greater than 90% (P less than 0.001) decrease in plasma prolactin level was observed in naltrexone-treated rats compared to the untreated animals. Rats subjected to restraint stress showed a 15% (P less than 0.001) decrease in weight gain at the end of the experiment (18 weeks). Neither restraint stress nor naltrexone administration affected the caloric intake of rats during this period. Thus, we believe that restraint stress facilitates DMBA-induced mammary tumors by releasing beta-endorphin and prolactin, and naltrexone shows a beneficial effect by opposing the effect of beta-endorphin on prolactin release in the stressed animals.

Preproenkephalin mRNA and methionine-enkephalin increase in mouse striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment.

Dopaminergic neurons that project to the striatum from the substantia nigra are thought to modulate methionine-enkephalin (Met-Enk) metabolism in the striatum. We administered a dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that produces a moderate depletion of dopamine in striatum, about 50%, without overt motor deficits, and found that Met-Enk-like immunoreactivity and preproenkephalin mRNA content increased in the tissue. Pretreatment with the monoamine oxidase B inhibitor deprenyl or the dopamine transport blocker nomifensine prevented these changes, suggesting that the changes were related to the partial loss of dopaminergic neurons rather than to MPTP. Moreover, administering GM1 ganglioside, which partially restores the MPTP-induced dopaminergic deficit, partially corrected the Met-Enk changes in the striatum as well. These findings are consistent with the hypothesis that dopaminergic input to the striatum, in part, modulates Met-Enk metabolism. Moreover, they show that moderate nigrostriatal lesions are sufficient to elevate Met-Enk and preproenkephalin mRNA contents and that restoration of dopaminergic function, as in our studies with GM1 ganglioside, restores the content of Met-Enk.